Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes

Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle’s host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.


Introduction
Many interactions between insects and their host plants are known to be mediated by volatile organic compounds. In contrast, volatile signals between herbivorous insects and their symbiotic microbes have been less studied, aside from a few well-known examples including ambrosia beetles, termites, and the vinegar fly Drosophila melanogaster [1][2][3][4]. Yet, such signals could be as critical for insect fitness as their response to host plant cues. In some insect-microbe symbioses, microbes transform host plant metabolites creating volatile signals that are used by insects for food or brood site selection [3][4][5][6]. For example, yeasts vectored by D. melanogaster metabolize dietary phenolic antioxidants and release volatile phenolics that attract both larvae and adults to feed on antioxidant-rich foods [7]. Nevertheless, there is still little information about how microbial transformation of host plant chemicals influences insect-microbe symbioses, and whether the resulting metabolites represent honest signals of partner benefits.
Microbial symbioses are especially characteristic of wood-boring insects such as bark and ambrosia beetles. Bark beetles have captured much attention recently because of their largescale outbreaks in many parts of the world. In Europe, for example, the Eurasian spruce bark beetle (Ips typographus) has killed millions of hectares of spruce stands as a result of major population bursts, likely in response to global warming and management practices that increase forest vulnerability to epidemic outbreaks [8][9][10][11][12]. This bark beetle feeds and reproduces in the phloem tissue of trees, which contains high levels of terpene and phenolic defense chemicals [8,13]. This insect overcomes its unfavorable environment by mass attacks and by introducing a suite of microbes into the host tree, including ectosymbiotic ophiostomatoid fungi, primarily Grosmannia penicillata, Leptographium europhioides, Endoconidiophora polonica, and Ophiostoma bicolor that cause blue staining of infected wood [14][15][16][17][18]. The ecological relationships between these fungal symbionts and I. typographus have not been extensively studied. Although they grow outside the insect, as necrotrophic fungi, they may serve as mutualists by exhausting host tree defenses, metabolizing host defense compounds, and providing nutritional benefits to larvae and adults [19][20][21]. However, some ophiostomatoid fungal associates of bark beetles may also act as commensals or antagonists to beetle brood development [22].
Conifer oleoresins are a formidable defense against insects and pathogens, as they can poison and physically entrap invaders [23][24][25][26]. However, the volatile fraction of the resin, especially the monoterpenes, also plays a central role in the colonization of host trees by bark beetles [24,27,28]. After locating a suitable tree, pioneer male I. typographus oxidize the dominant host monoterpene α-pinene to cis-verbenol, which is used as an aggregation pheromone in combination with the de novo produced 2-methyl-3-buten-2-ol to attract conspecifics for a mass attack [29][30][31]. In addition to bark beetle pheromones, several other oxygenated monoterpenes such as terpinen-4-ol, camphor, trans-4-thujanol, and borneol have also been detected at the entrance holes of I. typographus galleries [32][33][34][35]. Additionally, the phloem colonized by ophiostomatoid fungi around these galleries also produces large amounts of oxygenated monoterpenes compared to galleries without evident fungal growth [33]. Only a few studies have investigated the changes in the oleoresin composition of conifers within the necrotic lesions caused by fungi [36,37]. While these studies showed that fungal infection changed the composition of oleoresins, the presence of oxygenated monoterpenes and their ecological roles were not investigated. In our previous work, we showed that I. typographus utilizes de novo synthesized fungal volatiles to maintain its association with specific beneficial symbionts and also to avoid saprophytes [38]. However, it is unknown which volatiles are produced by these fungi when they colonize their native substrate i.e., the phloem and sapwood of the tree.
In this study, we investigated the volatile compounds emitted when fungal symbionts of I. typographus infect the excised bark of their Norway spruce (Picea abies) host trees. We show that these fungi alter the volatile monoterpene composition of spruce bark and demonstrate, using single sensillum recordings (SSRs), that adult I. typographus can perceive the fungal-produced monoterpenes and are attracted to these compounds in behavioral bioassays. Our results imply that bark beetles respond to fungal symbiont biotransformation products of host tree metabolites and employ them to identify suitable sites for feeding and breeding.

Ips typographus is attracted to volatiles from fungal symbionts on a spruce bark medium
Adult I. typographus beetles were strongly attracted to volatiles emitted by their symbiotic fungus G. penicillata grown on both potato dextrose agar (PDA) or spruce bark agar (SBA) compared to the respective fungus-free agar controls in laboratory trap bioassays (Fig 1) (PDA, z = 3.34, p = 0.001; SBA, z = 2.83, p = 0.005, Wilcoxon's test). Further, bark beetles showed a stronger attraction towards G. penicillata grown on SBA over the same fungus grown on PDA ( Fig 1D) (z = 4.28, p < 0.001, Wilcoxon's test). Volatiles from several other bark beetle fungal symbionts, such as E. polonica and L. europhioides, grown on SBA were also attractive to adult beetles, although not all bark beetle-associated fungi, nor an antagonistic, nonsymbiotic saprophyte (Trichoderma sp.), tested in this way emitted attractive volatile blends (S1 Fig) [39].

Symbiotic fungi alter the volatile profile of the bark
The headspace volatiles of P. abies bark inoculated with I. typographus fungal symbionts were distinct from those of fungus-free bark 4 d after inoculation when analyzed using gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) (S2-S7 Tables). For G. penicillata-inoculated bark, PCA analysis revealed that nearly 68% of the variation in the volatile profiles was explained by the first two principal components (Fig 2A).
The monoterpene metabolites of G. penicillata grown on either (−)-α-pinene-or (−)-βpinene-enriched agar were similar, with the oxygenated monoterpene terpinen-4-ol being the major product from both spruce precursors (Fig 3B). Other oxygenated monoterpenes formed by G. penicillata in the presence of (−)-α-pinene and (−)-β-pinene included (+)-isopinocamphone, (+)-trans-4-thujanol and α-terpineol, with (−)-verbenone being detected only from (−)-α-pinene (S5 and S6 Figs). From (−)-bornyl acetate, the dominant oxygenated  Table. (B) Changes in volatile emission profiles of spruce bark due to G. penicillata infection over an 18-d time course. Compounds are classified into six groups by their chemical structures (n = 3 to 5). Each circle displays the relative amounts of each class of compounds at a single time point and does not indicate changes in the total amounts of volatiles. Complete volatile emission data by compound and time point for G. penicillata and other symbionts are given in S3-S6 Tables. (C, D) Emission of specific monoterpenes from fresh spruce bark inoculated with G. penicillata at 4 days post-inoculation. Identified compounds were classified into monoterpene hydrocarbons (C) and oxygenated monoterpenes (D). The individual compounds are stacked within a single bar representing the total emission. Significant differences in the total emission levels induced by G. penicillata are denoted by asterisks above the bars (n = 4, Welch's t test with ns = not significant � P < 0.05, �� P < 0.01, ��� P < 0.001). The numbers denote the identities of the compounds in the stacked bars. Complete volatile emission data are given in S2 Table. (E) Emission rate of major oxygenated monoterpenes from spruce bark inoculated with G. penicillata 4 d post-inoculation. Asterisks indicate significant differences between the spruce bark-inoculated G. penicillata and the fungus-free control (Welch's t test). The data underlying this Figure  and their production coincided with the decrease of the precursor (Fig 3A). Similar results were obtained for the three other fungal symbionts tested (S4-S8 Figs).
The volatile blend of the co-occurring, but nonbeneficial fungus, Trichoderma sp., produced from a synthetic spruce monoterpene mixture (S8 Table) was distinct from that of G. penicillata (S9 Fig). Trichoderma sp. produced mainly the oxygenated monoterpenes, (−)-borneol and (−)-verbenone, while G. penicillata produced mainly (−)-camphor and terpinen-4-ol. These results collectively show that symbiotic fungi can alter the volatile profile of spruce bark by increasing the emission of oxygenated monoterpenes, and the emission profile of symbiotic fungi is distinct from that of a co-occurring fungal saprophyte.

Elimination of symbiotic ophiostomatoid fungi from I. typographus reduces the production of oxygenated monoterpenes in bark beetle galleries
To determine the primary source of oxygenated monoterpenes in beetle galleries, unsterilized pupae were reared into fungus-free mature adults using a diet fortified with the fungistatic agent sodium benzoate (S1 Method). Sterility tests and scanning electron microscopy of beetle body parts revealed that beetles reared on this diet were free from fungi and the elytral pits were devoid of fungal spores and yeasts (S15C Fig and S9 Table), although bacteria were abundant. In comparison, elytral pits of beetles reared on diet without sodium benzoate contained abundant spore masses, with structural similarity to those of O. bicolor and yeasts (S15B Fig  and S9 Table) [40]. Reinoculation of fungus-free beetles with G. penicillata resulted in acquisition of G. penicillata spores in the elytral pits (S15D Fig). Next, we quantified the amount of oxygenated monoterpenes present in the bark galleries constructed by untreated, fungus-free, and G. penicillata-reinoculated beetles separately using GC-MS analyses of gallery extracts. The concentration of (−)-camphor was significantly higher in the galleries of reinoculated beetles than in bark samples without beetles at all (F (3,23) = 15.6, p < 0.001, ANOVA), while the concentration of terpinen-4-ol was significantly higher in untreated beetles than in both fungus-free beetles or in bark samples without beetles (H (3) = 18.6, p < 0.001, Kruskal-Wallis) (Fig 4B). On the other hand, the concentrations of these two oxygenated monoterpenes were similar when comparing galleries of G. penicillatareinoculated beetles to those of untreated beetles. For (+)-isopinocamphone, the concentration in galleries constructed by untreated beetles was significantly higher compared to bark samples without beetle galleries, but similar to that in galleries constructed by fungus-free and G. penicillata-reinoculated beetles (F (3,23) = 5.7, p = 0.005, ANOVA) ( Fig 4B). On the other hand, the concentration of (−)-borneol in fungus-free galleries was significantly higher compared to that in bark samples without galleries and was similar to that in the galleries of G. penicillatareinoculated and untreated beetles (F (3,23) = 5.3, p = 0.006, ANOVA) ( Fig 4B). There were no significant differences in the concentrations of pinocamphone, α-terpineol, and (−)-verbenone among treatments.
Analysis of the microbes present in each treatment revealed that galleries constructed by fungus-free beetles were indeed free from symbiotic ophiostomatoid fungi and yeasts but contained many saprophytes and (or) endophytes and bacteria (S10 Table). Microbes present in the galleries of untreated and G. penicillata-reinoculated beetles were similar to those found on beetles that were used to infest the bark (S9 and S10 Tables).

Ips typographus detects oxygenated monoterpenes through specialized olfactory sensory neurons (OSNs) in their antennae
To test if I. typographus antennal olfactory sensilla contain OSNs that detect the biotransformation products of monoterpenes, we challenged 231 olfactory sensilla with a test panel comprising 92 ecologically relevant compounds diluted in paraffin oil (1 μg μl −1 ) using single cell recordings (S1 Table). Only 23 (approximately 10%) of the sensilla housed neurons that did not respond to any of the compounds from the odor panel, although their OSNs showed spontaneous firing. We obtained odor-evoked responses with strong excitation (>120 Hz) from 198 OSNs and weak excitation (<50 Hz) from 10 OSNs, allowing the grouping of these neurons into different classes based on their response profiles. From initial screening experiments at a 10-μg dose on filter paper (to determine the maximum receptive range of OSNs), we identified 20 classes of OSNs. Three OSN classes responded primarily to fungal-produced oxygenated monoterpenes. We also identified neurons belonging to previously described OSN classes tuned to pheromones, host tree volatiles, and nonhost odorants [41] that are not further considered here.
A few previously characterized OSN classes for host tree monoterpenes, including the classes with primary responses to α-pinene, p-cymene, and Δ3-carene, respectively [41], showed varying secondary responses to some of the fungal-derived compounds tested here for the first time. For example, the α-pinene OSN class responded also to
Addition of another host tree monoterpene, (−)-bornyl acetate, to fungal growth medium at 0.05 mg g −1 (in the range of natural concentrations in P. abies bark) and 0.5 mg g −1 resulted in strong attraction of I. typographus adults towards G. penicillata when tested against a fungus-free control after 4-d incubation (Fig 6C, left panel) (0.05 mg g −1 , z = 3.31, p = 0.001; 0.5 mg g −1 , z = 3.21, p = 0.001, Wilcoxon's test). This attraction was correlated with the presence of fungal biotransformation products from (−)-bornyl acetate ( Fig 6C, right panel). The major biotransformation product that formed in this period ( Fig 6C) was camphor, which was significantly more attractive to adult beetles at a 100-μg dose than the mineral oil control ( Fig 6D) (z = 2.58, p = 0.01, Wilcoxon's test). However, camphor had no effect at larger or smaller doses. Adult beetles preferred G. penicillata grown on unenriched medium against G. penicillata grown on medium enriched with a high amount of (−)-bornyl acetate (0.5 mg g −1 ) ( Fig 6F) (z = 2.12, p = 0.03, Wilcoxon's test). By contrast, in the absence of fungus, beetles did not discriminate between diet enriched with monoterpenes and diet without monoterpenes (S11 Fig). Thus, I. typographus is attracted to the major oxygenated fungal metabolite of (−)-bornyl acetate but is not attracted and even repelled by (−)-bornyl acetate itself.

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Conifer bark beetles detect volatiles of fungal symbionts produced from host tree resin monoterpenes

Symbiotic fungi increase the tunneling of adult I. typographus
The presence of a symbiotic fungus increased the tunneling of adult I. typographus beetles in medium after 48 h. Multiple logistic regression analysis revealed that successful tunneling odds in (−)-bornyl acetate-amended medium were significantly influenced by the fungus when the beetle sex and the monoterpene treatment remained constant (Table 1) (β = 4.98, χ 2 = 20.99, p < 0.001). The presence of the fungus increased the tunneling probability by 99% compared to in the absence of the fungus. Additionally, males had significantly lower tunneling success compared to females (Table 1) (β = −1.72, χ 2 = 8.78, p = 0.003) with the tunneling probability for males being 15.2% lower than for females. In (−)-β-pinene-amended medium, only the presence of the fungus significantly influenced the tunneling probability of beetles (Table 1) (β = 2.65, χ 2 = 20.45, p < 0.001), with an increase of 93% compared to in the absence of the fungus. Similarly, in (−)-α-pinene-amended medium, only the presence of the fungus significantly influenced the tunneling probability of beetles (Table 1) (β = 2.02, χ 2 = 9.39, p = 0.002), with an increase of 88% compared to the absence of the fungus. Addition of the three monoterpenes without fungus did not have any effect on the tunneling behavior of adult beetles.
The growth of G. penicillata on monoterpene-enriched media resulted in significantly longer beetle tunnels than in the other treatments ( Fig 8B-8D ANOVA, Tukey's test)). However, there were no other significant interactions between monoterpenes and G. penicillata affecting tunneling, i.e., no other differences in tunnel lengths when comparing fungus alone versus fungus plus monoterpenes except for the treatment with (−)-β-pinene (Fig 8B-8D, Tukey's test). The sex of the beetle and the interaction of sex with other treatments also had no effect on the tunnel length.

Discussion
The successful attack of bark beetles on their host trees is invariably associated with free-living fungal symbionts. These symbionts may detoxify the terpene-rich defensive resin of the host tree, hasten host tree death, provide nutritional benefits, and increase resistance to pathogens [16,17,21]. Here, we document the ability of I. typographus fungal symbionts to metabolize host tree monoterpenes to oxygenated derivatives based on experiments in which we cultured fungi with and without host tree monoterpenes. Several oxygenated monoterpenes have been previously identified as volatiles released from trees that were attacked by I. typographus [32,33,35]. We showed that these are fungal metabolites that become dominant components of the volatile profile making up over half of the total bark volatiles emitted 12 d following infection. Bark beetle OSNs can detect many of these oxygenated monoterpenes, and some of these compounds attract adult beetles to diets inoculated with fungal symbionts. We hypothesize that oxygenated monoterpenes may assist adult beetles in locating suitable breeding and feeding sites [34,45].

Bark beetle symbiotic fungi metabolize host tree monoterpenes to oxidized derivatives
Fungi are well known to metabolize monoterpenes as well as many other chemical compounds they encounter in their natural environments [46,47]. In particular, fungi associated with conifer trees are reported to transform different types of resin monoterpenes to oxidized derivatives [48,49]. Previous literature describes this capability for a symbiotic fungus of the North American bark beetle Dendroctonus ponderosae [50]. Here, we showed that fungal symbionts of the European spruce bark beetle I. typographus also oxidize monoterpenes of their host trees by adding monoterpene standards to the culture medium and by growing symbionts on spruce bark with natural levels of monoterpenes.
Oxidation of monoterpenes can be considered a detoxification process since these lipophilic compounds have antifungal properties [49,50], and oxidation may facilitate excretion by increasing polarity. However, individual oxidative transformations may not necessarily decrease toxicity but could serve as entry points to reaction cascades that allow monoterpenes to be degraded and used as carbon sources for intermediary metabolism [50,51]. Here, we only searched for the volatile monoterpene metabolites of fungal symbionts and so cannot speculate on whether or not monoterpenes are employed as carbon sources.
Although we did not use isotopically labeled substances to confirm that fungal symbionts metabolize host tree monoterpenes to oxygenated derivatives, the de novo synthesis of oxygenated cyclic monoterpenes by these fungi can be almost ruled out. In previous work, we collected volatiles from G. penicillata and the other I. typographus-associated fungi studied here growing under many conditions without spruce bark or supplemental monoterpenes [38]. While we detected a large range of volatile compounds, we never found any trace of oxidized cyclic monoterpenes like those reported here, which all have the bornane, p-menthane, pinane, or thujane carbon skeletons characteristic of spruce monoterpenes. Some fungi do produce cyclic monoterpenes of their own, but none have yet been reported to synthesize the variety of cyclic skeletons produced by conifers, and so are very likely to obtain their precursors for oxygenated monoterpenes from conifer trees.

Oxygenated monoterpenes are widespread volatile cues for tree-feeding insects
Various forest insects that are associated with fungal symbionts, such as bark beetles, ambrosia beetles, and wood wasps, live in host trees producing large quantities of monoterpene volatiles. These insects are often attracted to their fungal symbionts through volatiles [2,38,52], and, hence, fungus-produced monoterpene metabolites could be critical components of the attractive volatile blends. For bark beetles, oxygenated monoterpenes derived from either the beetles, fungi, or host trees are already known to play important roles in their life history [53]. During Table 1. Multiple logistic regression analysis predicting the odds of adult I. typographus tunneling into media enriched in different monoterpenes with and without G. penicillata in a no-choice assay (see also Fig 6A). The data underlying this

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mass attacks, the host-derived oxygenated monoterpene cis-verbenol acts together with 2-methyl-3-buten-2-ol as an aggregation pheromone for I. typographus to promote mass attack on individual trees [28,54]. Beetles also utilize oxygenated monoterpenes to restrict the density of attack. Microbes lining the gallery walls or living in the beetle gut oxidize cis-and trans-verbenol into verbenone, which inhibits the attraction of both sexes to densely colonized trees [55][56][57]. In addition, mated male I. typographus produce ipsenol and ipsdienol, of which ipsenol acts as an anti-attractant [57]. Furthermore, an oxygenated monoterpene from host trees, 1,8-cineole, which is produced in higher amounts in resistant or MeJA-primed trees, inhibits

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attraction of beetles to their pheromones [42,58,59]. Oxygenated monoterpenes are also used as reliable cues by parasitoids of bark beetles to locate their prey [35,60].
Here, we discovered that a blend of oxygenated monoterpenes emitted by I. typographusassociated fungi growing on agar medium amended with spruce bark or specific spruce monoterpenes is highly correlated with beetle attraction (Fig 6A and 6C). These same fungi were shown in a previous study [38] to produce mixtures of non-terpene aliphatic and aromatic volatiles when grown without sources of monoterpenes, and a blend of these aliphatic and aromatic compounds attracted newly emerged (callow) adult I. typographus [38]. These nonterpene compounds were also detected in the present study as major components of the volatile blend at later phases of fungal growth (S3-S7 Tables). Although we focused principally on the symbiont G. penicillata in our work, volatiles from other fungal symbionts were also investigated. L. europhioides and E. polonica emitted volatiles on SBA that were also attractive to adult I. typographus (S1 Fig), but the volatiles of O. bicolor and saprophytes such as O. piceae and Trichoderma sp. were not attractive.
The production of oxygenated monoterpenes by other microbes coinhabiting I. typographus galleries, such as fungal saprophytes, yeasts, and bacteria, has been reported before, but these microorganisms produce different metabolites of host tree monoterpenes (or the same metabolites in different ratios) [61][62][63] than those produced by symbiotic fungi as reported here. In addition, the blend of oxygenated monoterpenes emitted by I. typographus alone was distinct from that emitted by the fungal symbionts [64] (S12 Fig). Since oxygenated monoterpenes are major components of the volatile profiles of I. typographus fungal symbionts that are attractive to beetles (S2-S7 Tables and S9 Fig), it can be suggested that the oxygenated monoterpenes themselves are important in I. typographus attraction. Bioassays of individual oxygenated monoterpenes showed that two were attractive to beetles at specific concentrations, and two had no effect (Fig 6B and 6D). I. typographus was recently reported to be attracted to the fungi associated with the North American spruce bark beetle D. rufipennis when attacking a different species of spruce than I. typographus normally does in its home range. This attraction could also be due to oxygenated monoterpene profiles, although it is unclear if an association with these fungi would be beneficial to I. typographus [65].

Oxygenated monoterpenes signal the presence of fungi to I. typographus and may modulate beetle colonization
The first chemical signals reported to mediate bark beetle colonization of their hosts were aggregation pheromones. Yet even in the presence of these pheromones, a large proportion of aggregating beetles that land on trees leave without tunneling into the bark [45,66,67]. This suggests that other cues may be needed to induce beetles to stay and bore into the bark. Indeed, bark beetles have been shown to respond to visual and chemical cues from host and nonhost species when selecting trees for colonization [28,38,44,[68][69][70]. Based on our results, fungusproduced oxygenated monoterpenes might also belong to the list of colonization cues. For bark beetles, fungal metabolites can serve as indicators of host tree sectors where their fungal symbionts are already established. These compounds could also provide evidence for the ongoing metabolism of host tree defenses or signal that tree defenses have been overwhelmed, which could improve the success of bark beetle colonization.
Fungal volatiles also enhance the attraction of I. typographus to aggregation pheromones. Female I. typographus is known to use the aggregation pheromone mixture (2-methyl-3-buten-2-ol and cis-verbenol) to locate trees suitable for mating and oviposition [45,71].
Here, we showed that female I. typographus were more attracted at short range to a combination of the pheromone mixture plus fungal volatiles than to the pheromone mixture alone.
Similarly, a recent study reported that the presence of fungal volatiles increased the attraction of dispersing beetles to their pheromones in the field compared to traps with pheromones only [72]. Oxygenated monoterpenes and other fungal volatiles could provide information about the presence of fungal symbionts, which promote the successful development of their offspring. Similarly, the pheromone component cis-verbenol, itself an oxygenated monoterpene produced by I. typographus from the host tree precursor α-pinene [73], provides information about the presence of other beetles, especially males. The lack of response of males to pheromones in our experiments is not unexpected, as male I. typographus have been reported to be less responsive than females to high doses of pheromones in walking bioassays [71,74]. This behavior may help them avoid dense colonies of male conspecifics within a tree to reduce competition for mates and food.
Depending on how fast symbiotic fungi colonize the tree bark, fungal cues could benefit both epidemic and endemic beetle populations. During the endemic phase, beetles generally colonize stressed trees in a gradual fashion over months [66], and in this case, volatiles emitted from fungal symbionts introduced into the tree by early colonizers, in combination with beetle pheromones, could increase the probability for conspecifics to locate attacked trees, present at low densities in spruce stands. In contrast, during epidemics, vigorous trees are typically colonized by large numbers of bark beetles within a few days, which overwhelms their defenses. Since ophiostomatoid fungi have been reported to emit volatiles within 1 d following the infection of detached bark plugs in the laboratory [75,76], these chemicals might function in attraction during epidemics as well. However, during rapid natural attacks, it is unclear if fungi can colonize a tree sufficiently to emit behaviorally active volatiles. More research in this area is needed.
The oxygenated metabolites of host monoterpenes produced by fungal symbionts may not only attract bark beetles, but also stimulate them to tunnel in a fungus-colonized medium. I. typographus tunneled more in a diet with G. penicillata and β-pinene than with either fungus or monoterpene alone, perhaps because the fungus metabolized β-pinene to behaviorally active metabolites. However, this effect was not seen for α-pinene or bornyl acetate. Interestingly, both sexes showed similar tunneling preferences to fungi and monoterpenes in contrast to their different response to fungi and pheromones. The lack of sex-specific differences in tunneling could be due to the fact that the nutritional advantage of feeding on fungus-colonized spruce bark is beneficial to both male and female adults [17,18]. No other bioassays performed in this study showed sex-specific differences.
The proportion of oxygenated monoterpenes to total monoterpenes in the volatile blend of G. penicillata increased over the time course studied to nearly 50% at 12 d and nearly 70% at 18 d post-inoculation, a trend also observed for L. europhioides and O. bicolor. Thus, higher proportions of oxygenated monoterpenes may indicate older fungal infection sites and, hence, older beetle invasion sites that may be less attractive to newly arriving beetles due to crowding. The lack of attraction and even repellency of certain individual oxygenated monoterpenes, as seen in laboratory bioassays in our own and in previous studies [77] is consistent with this interpretation. Among the oxygenated monoterpenes already reported to inhibit I. typographus attraction, verbenone, produced by microbial or autooxidation of the pheromone cis-verbenol (or by direct oxidation of α-pinene), reduces aggregation of I. typographus in later phases of the attack on the tree [57,63,78].
Oxygenated monoterpenes are signals not only for bark beetles, but also for their natural enemies. Both beetle predators and parasitoids employ these compounds and other volatiles to locate bark beetle larvae hidden under the bark [79,80]. Specifically, a three-component blend comprising camphor, isopinocamphone, and terpinen-4-ol, all fungal metabolites of host tree monoterpene hydrocarbons, was reported to attract a coleopteran predator and several hymenopteran parasitoids of I. typographus in the presence of host tree background signals [60,81,82]. Furthermore, the bark beetle predator, Thanasimus formicarius, possesses OSNs to detect oxygenated monoterpenes such as camphor and pinocamphone as well as several additional OSN classes detecting various oxygenated bark beetle pheromone compounds [83]. Thus, any benefit to the beetle arising from oxygenated monoterpene production by its symbiotic fungi may come at the cost of revealing its presence to natural enemies that employ these same volatiles to locate bark beetles.

Formation of oxygenated derivatives may reduce monoterpene toxicity for I. typographus
The conversion of host tree monoterpene defenses by symbiotic fungi to oxygenated products may alleviate toxicity to bark beetles. Terpene-rich resins are a general defense of P. abies and other conifers against herbivores and pathogens [84][85][86]. Thus, it is not surprising that monoterpenes have exhibited toxicity to bark beetles in many studies [87][88][89]. Monoterpene hydrocarbons, such as α-pinene, are typically more toxic to beetles than host tree-produced oxygenated monoterpenes, such as bornyl acetate [90]. Hence, the oxidative transformations carried out by fungal symbionts described in this study could reduce toxicity to I. typographus through conversion to less poisonous derivatives. Such detoxification of host tree defenses could represent a significant benefit of fungal symbionts [91].
By contrast, oxygenated monoterpenes may be more toxic to fungi compared to monoterpene hydrocarbons [48,49]. Thus, the initial oxidation of monoterpene hydrocarbons may not constitute a detoxification unless it is a step towards further metabolism. The potential toxicity of oxygenated monoterpenes may explain why these substances are degraded further after initial oxidation by fungi specialized on conifers such as Heterobasidion parviporum and Seridium cardinale [48,49]. Another class of terpenes present in P. abies bark and other conifers host trees of bark beetles are diterpene resin acids, which were reported to be toxic to associated fungi in a North American bark beetle system [92].

Other sources of oxygenated monoterpenes in spruce bark beetle interactions
Oxygenated monoterpenes emitted from trees attacked by I. typographus may arise from sources other than fungal symbionts. The host tree P. abies itself synthesizes large amounts of bornyl acetate [93] and small amounts of 1,8-cineole [94]. In these compounds, the oxygen functions are incorporated during biosynthesis from basic precursors, whereas the products from fungal symbionts are presumably formed by oxidative modification of a previously formed monoterpene hydrocarbon skeleton. The compound trans-4-thujanol belongs to the latter group. We identified it as a G. penicillata metabolite of α-and β-pinene, but trans-4-thujanol may also be synthesized by the tree, although at low levels in P. abies bark [77]. As an alternative, this and other oxygenated monoterpenes could be produced via autoxidation of monoterpene hydrocarbons [95]. The oxidation of monoterpenes upon exposure to air could explain the release of low but readily detectible amounts of these compounds from uninfected control bark plugs in our and other studies. In the field, oxygenated monoterpenes other than bornyl acetate and 1,8-cineole have been detected from damaged P. abies trees when monoterpenes were exposed to air [96,97]. However, in the present study, the emission rate from uninfected bark plugs was much lower than from fungus-infected plugs, suggesting that microbial metabolism is a much more significant source of oxygenated monoterpenes than autoxidation (S9 Fig) [95]. Nevertheless, the tree itself cannot be ruled out as a source of any of the detected oxygenated monoterpenes, since P. abies cell suspension cultures have been reported to oxidize added monoterpenes [94,95]. If P. abies produced monoterpenes as a defense response only when infected by fungi, it might be difficult to determine which organism was producing these compounds-the tree or the fungus-without identifying specific biosynthetic genes in one of the two genomes. More research is needed to assess the contribution of host trees to the oxygenated monoterpenes emitted in response to beetle tunneling or fungal infection.
Among microbial sources of oxygenated monoterpenes are several yeast species including Hansenula holstii, H. capsulata, and Candida nitratophila, which were isolated from I. typographus and produce terpinen-4-ol, α-terpineol, borneol, and trans-pinocarveol when grown in phloem medium or in α-pinene-supplemented medium [63]. In addition, several bacterial symbionts of bark beetles are capable of metabolizing monoterpenes into oxygenated derivatives, such as verbenols and verbenone [62]. Some bacteria coinhabit the beetle gallery together with ophiostomatoid fungi, but it is not known if these bacteria induce the production of oxygenated monoterpenes, either by producing them themselves or by interacting with fungi to form these compounds [98]. Another bark beetle species, Polygraphus poligraphus, which is sometimes found together with I. typographus, was shown to emit large amounts of terpinen-4-ol [99,100]. Intermediate amounts of α-terpineol, cis-and trans-4-thujanol were also identified from the hindgut as well as the entrance holes of this beetle's gallery and could be formed by microorganisms associated with this beetle species from host tree monoterpenes or by the beetle itself. Our experiments with fungus-free I. typographus, however, demonstrated that live beetles themselves did not produce high concentrations of any of the fungal biotransformation products identified in this study. Instead, beetles formed a range of other oxidation products (S12 Fig) that have been identified in previous studies [64,101]. In summary, our study brings together several lines of evidence showing that the oxygenated derivatives of spruce resin monoterpenes, which appear during I. typographus attack, are produced mainly by the metabolism of symbiotic fungi. The fungal origin of these compounds had been suggested previously [32] but not supported by experimental evidence until now.
High selectivity of I. typographus olfactory neurons to oxygenated monoterpenes suggest their role in detecting symbiotic fungi I. typographus possesses several classes of OSNs that were shown to detect the oxygenated monoterpenes produced by their fungal symbionts with notable specificity. For example, the isopinocamphone OSN class showed high specificity towards several monoterpene ketones produced by fungal symbionts, including (+)-and (−)-isopinocamphone, (+)-and (−)-pinocamphone, camphor, and pinocarvone, but not to monoterpene alcohols such as borneol and trans-pinocarveol. The absence of any response to monoterpene hydrocarbons indicates that this OSN is not tuned to detect the host tree itself, but rather organisms metabolizing the major host monoterpenes. The isopinocamphone OSN responded similarly as one recently reported OSN class from I. typographus that responded best to pinocarvone and camphor (OSN class named Pcn; isopinocamphone and pinocamphone were not tested) [97]. Our work shows that (+)-isopinocamphone is the primary ligand of this OSN class, based on its greater activity than the other active compounds. In addition, the response profile of this OSN class matches very well with that of the I. typographus odorant receptor 29 (ItypOR29), which recently was characterized in the oocytes of the African clawed frog, Xenopus laevis [102].
Likewise, we showed that a previously described verbenone-sensitive OSN class [41] also responds to cis-and trans-verbenol as well as α-and β-isophorone, compounds that are believed to arise from bark beetle metabolism of host tree terpenes [64]. Verbenone is produced from the verbenols by microbes that colonize gallery walls and beetle guts. Therefore, this OSN class appears to be tuned to signals from various ecological sources providing information on bark beetle density as well as microbial establishment [45,56,96]. Since, as discussed above, individual oxygenated monoterpenes arise from different organisms, including fungi, other microbes, the host tree, and the beetles themselves, the presence of OSNs responding to different oxygenated monoterpenes may help the beetle identify the various life forms it encounters.
Another OSN class responded most sensitively to the monoterpene alcohol trans-4-thujanol, a fungal symbiont metabolite of α-and β-pinene. This OSN also responded to the fungal metabolites terpinen-4-ol and α-terpineol, as well as C 8 alcohols, but only at the highest doses tested, which extends prior results for this OSN [97] to other compounds from our greatly expanded test odor panel. Strong electroantennographic activity in I. typographus in response to these oxygenated monoterpenes has also been reported [96,97], and the response spectrum of this OSN class matches well with that of the receptor ItypOR23, which is evolutionarily related to the receptor ItypOR29 that detects isopinocamphone [102]. The specific responses of I. typographus bark beetles to oxygenated monoterpenes may arise not only from the selectivity of the OSN classes, but also due to further processing of the odor signal in the antennal lobe and other higher centers of the brain responsible for sensory integration (mushroom bodies and lateral horns) [103]. Moreover, since some of the oxygenated monoterpenes elicited secondary responses from other OSN classes, bark beetles like other insects may process olfactory information through combinatorial codes [70], which could lead to very specific responses to different compounds or mixtures.

Conclusions
We have shown that fungal ectosymbionts vectored by I. typographus increased the emission of oxygenated monoterpenes from spruce bark due to fungal metabolism of host tree monoterpenes. These oxygenated volatiles were detected by several classes of I. typographus OSNs; some volatiles attracted beetles at specific concentrations, while others had no effect. Oxygenated monoterpenes appear to signal the presence of established fungi, but their effects on beetles are context-dependent and could vary with the physiological status of the fungi, the age, sex, and density of beetles, and the stage of attack [38,76]. Moreover, the ecological roles we have proposed for these oxygenated monoterpenes are based on laboratory assays with walking beetles, so studies under natural conditions are necessary to confirm our findings. These compounds may also be useful in integrated pest management strategies as attractants or repellents of bark beetles perhaps in combination with pheromones [104][105][106]. In this way, oxygenated monoterpenes and other microbial volatiles represent a rich source of untapped insect semiochemicals that can be exploited for protecting forests from devastating pest species such as I. typographus.

Fungal strains and growth medium
The fungal strains used in this study have been previously described [38] (listed in S1 Table). In order to obtain spores from fungi, freshly inoculated PDA plates were incubated at room temperature for 15 to 20 d until the mycelium was old and dark. After 20 d, plates were kept briefly at 4˚C to induce sporulation. Four to six 1 cm diameter mycelium plugs were removed from each plate and inoculated into 20 mL potato dextrose broth and incubated at 25˚C at 150 rpm for 4 days. Once the broth was turbid, the spores were filtered using a 40-μm cell strainer (Greiner Bio-One, Frickenhausen, Germany), and the filtrate was spun down at 4,200 rcf for 10 min to precipitate the spores. The supernatant was discarded, and the spore suspension was washed three times with autoclaved water and then stored at 4˚C until used. The spore suspension prepared using this method was viable for several months when stored at 4˚C.

Bark beetle rearing
Bark beetles were reared and stored in the laboratory as described [38]. The starting beetle culture was obtained from an infested tree in October 2017 near Jena, Thuringia, Germany. Beetles were reared throughout the year in the laboratory in freshly cut spruce logs (ca. 30 cm diameter × 50 cm height) placed in an environmental chamber set at 25˚C throughout the day, 65% relative humidity, and a photoperiod of 20 h per day. Beetles emerged from breeding logs after ca. 35 d and were collected manually. Emerged adults were sexed based on the bristle density on their pronotum [107] and stored separately in Falcon tubes lined with moist paper at 4˚C at least for a week before using them in bioassays. Adult beetles were used only once in bioassays.

Spruce bark diet
The outer bark of a freshly cut mature tree was scraped off gently using a drawing knife, and the inner bark (phloem) was carefully peeled off using a chisel and immediately stored at −80˚C. The bark was cut into small pieces and ground to a fine powder in vibratory micro mill (Pulverisette 0, Fritsch GmbH, Idar-Oberstein, Germany). The instrument was precooled with liquid nitrogen, and bark pieces were pulverized at an amplitude of 2.0 for ca. 10 min with addition of liquid nitrogen every 2 min to prevent thawing. The ground powder was stored in Falcon tubes at −80˚C until used for diet preparation. For preparing spruce bark diet, 7% powdered inner spruce phloem (w/v) was added to 4% Bactoagar (Roth) and heat sterilized at 121˚C for 20 min.

Identification and quantification of headspace volatiles of fungal symbionts
Collection and analysis of headspace volatiles for G. penicillata and Trichoderma sp. comparison (S9 Fig) is described in S2 Method. Norway spruce bark plugs of approximately 28 mm diameter were removed from a freshly felled tree in July 2017 and single bark plugs placed inside sterile 250 mL glass bottles for volatile collection. Before removing the bark plugs, the surface of the bark and the cork borer were sterilized by thorough spraying with 70% ethanol in a laminar hood. A 100-μL quantity of spore suspension (1 � 10 6 cells mL −1 ), prepared as described above, was added to the exposed section of the bark, and autoclaved water was added to the control treatment. Each treatment was replicated four times including the control. The glass bottle was secured tightly and incubated at 25˚C for 4 d. After 4 d, activated charcoal-filtered air was passed into the bottle inlet at the rate of 50 mL min −1 , and the outlet air was funneled through a SuperQ adsorbent filter (150 mg) for 4 h. Afterwards, the filters were eluted with 200 μL dichloromethane spiked with 10 ng μL −1 nonyl acetate (Sigma Aldrich) as an internal standard and stored at −20˚C. The spruce bark plugs were oven dried at 80˚C for 6 h after the experiment, and the dry weight was measured.
The eluted volatile samples were subjected to GC-MS analysis for identification and GC-FID analysis for quantification using an Agilent 6890 series GC (Agilent, Santa Clara, CA, USA) (injection, 1 μl splitless; flow, 2 ml min −1 ; temperature, 45 to 180˚C at 6˚C min −1 and then to 300˚C at 100˚C min −1 for 10 min) coupled either to an Agilent 5973 quadrupole mass selective detector (interface temperature 270˚C, quadrupole temperature 150˚C, source temperature 230˚C; electron energy 70 eV) or a flame ionization detector (FID, temp. 300˚C). The constituents were separated on a DB-5MS column (Agilent (30 m × 0.25 mm × 0.25 μm)), with He (MS) or H 2 (FID) as carrier gas. The GC-MS and GC-FID analyses used identical columns and temperature programs so that the chromatograms could be overlaid to facilitate identification and quantification. Peaks arising from the solvent and collection containers were identified by blank runs and excluded from the analysis. The identity of each peak was determined by comparing its mass spectra and retention times to those of reference libraries (NIST98 and Wiley275) and authentic standards. The amount of each compound was calculated from the peak area obtained from the FID detector relative to the internal standard and standardized to the spruce bark dry weight.

Time series headspace volatile collection
For time series volatile analysis, spruce bark plugs (10 mm diameter) were removed using a cork borer from a freshly felled spruce tree in October 2016. Each spruce bark plug was placed in a 15-mL clear glass vial (Supelco-Sigma-Aldrich), and 50-μL spore suspension (1 � 10 6 cells mL −1 ), prepared as described above, was added to treatment plugs while control plugs received sterile water. The headspace volatiles were captured on three polydimethylsiloxane (PDMS) sorbent silicone tubes (0.5 cm), which were hung in each glass vial using a manually crafted metal hook attached to the bottom of PTFE/silicone septa in the screw cap [108]. The headspace volatiles were collected from each treatment for 2 h at 4, 8, 12, and 18 d after inoculation. After sampling, silicone tubes were placed in 1.5 mL brown glass vials and stored at −20˚C until analysis.
Volatiles collected on PDMS tubes were analyzed using a GC-2010 plus gas chromatograph coupled to a MS-QP2010 quadrupole mass spectrometer equipped with a TD-20 thermal desorption unit (Shimadzu, Japan) and a GC Cryo-Trap filled with Tenax. A single tube was placed in a 89-mm glass thermal desorption tube and desorbed at a flow rate of 60 mL min −1 for 8 min at 200˚C under a stream of N 2 gas. The desorbed substances were focused on a cryogenic trap at −20˚C. The Tenax adsorbent was heated to 230˚C, and the analytes were injected using split mode (1:100) onto a Rtx-5MS GC column (30 m × 0.25 mm × 0.25 μm) with helium as carrier gas. Compounds were identified as above (1.5) from authentic standards and libraries, and the area of each peak obtained using GC-MS post run analysis software from Shimadzu. Peaks arising from contaminants from the solvent, medium, or bark plug container were identified by blank runs and excluded from the analysis. The PLS-DA plot in

Biotransformation of host tree compounds
Experiments were conducted in 9 cm Petri dishes containing 2% PDA supplemented with test solutions. The tested compounds were (−)-α-pinene, (+)-α-pinene, (−)-β-pinene, myrcene, γterpinene, terpinolene, sabinene, camphene, p-cymene, and (−)-bornyl acetate. Sources of these compounds are given in S1 Table. Compounds were added after dissolving in dimethyl sulfoxide (DMSO). These were then added into PDA (maintained at 55 to 57˚C using a water bath) to reach a concentration of 0.5 mg mL −1 before pouring into Petri dishes. A 5-mm agar plug containing a fungal colony was placed in the center of each dish and sealed tightly using several layers of Parafilm before incubation at 25˚C in darkness for 6 d. Each treatment was replicated four times, and for the control, the PDA contained only DMSO plus monoterpene. The headspace volatiles were collected after 4 d using three PDMS tubes, which were mounted on sterile metal wires and imbedded in PDA for 1 h and stored at −20˚C. The identification and quantification of compounds were conducted in the same way as reported for the time series (section 1.5). The headspace volatiles from fungus grown on PDA enriched with myrcene, γ-terpinene, terpinolene, camphene, and p-cymene did not yield detectable amounts of monoterpene transformation products on analysis.
To identify if symbiotic fungi can reduce the amount of monoterpenes in their substrate, fungi were grown on PDA enriched with 0.5 mg g −1 (−)-α-pinene, (+)-α-pinene, (−)-β-pinene, and (−)-bornyl acetate as described above. Control plates contained only DMSO and the tested monoterpene. After 4 d, three plugs of 6 mm diameter were removed, weighed, and transferred to 1.5 ml sterile glass vials. Agar plugs were homogenized using sterile plastic pestles and 1 ml hexane (extraction solvent) spiked with 10 ng μL −1 nonyl acetate was added and samples were vortexed for 30 s. Supernatants were transferred to new vials and stored at −20˚C until identification and quantification by GC-MS and GC-FID (1.4). Data analysis was identical to that reported above.

Electrophysiology
Laboratory reared adult beetles from the same German culture that were used in bioassays were used for electrophysiological SSRs using tungsten microelectrodes according to established methodology [38,41] with the equipment previously described from Syntech, Kirchzarten, Germany [114]. The odor panel comprising 92 compounds consisted of beetle pheromones, host tree, nonhost tree, and fungal compounds ( [38]; S2 Table). Both major and minor fungal volatiles identified during the chemical analysis were included in the odor panel. All odors were dissolved in odorless paraffin oil (w/v). SSR traces were analyzed as described [105] using Autospike 3.0 (Syntech). Males and females were initially screened for responses to the odor panel using a high-stimulus dose (10 μg on filter paper placed inside capped standard Pasteur pipette odor cartridges; [114]). OSN classes shown to primarily respond to fungus-derived oxygenated monoterpenes were subsequently studied in dose-response experiments with active stimuli diluted in 10-fold steps and tested from lowest to highest dose with the least active ligands tested first at each dose. To reduce variation due to odor depletion, stimulus cartridges were used for a maximum of 8 stimulations during screening and 2 stimulations during dose-response tests [115].

Trap bioassay
The arena used was described in [38]. In this apparatus, adult beetles had to make their choice through olfaction and not by contact cues. Fungi were inoculated on SBA-based diet and incubated at 25˚C for 4 d. With the help of a cork borer (10 mm diameter), bark plugs with or without fungus were inserted into circular plastic cups (1.8 cm height � 1.8 cm diameter) facing each other. Two beetles were placed at the center of each arena, and the olfactometer was placed inside a laminar flow cabinet in darkness. Each experiment was replicated at least 25 times on the same day with 2 beetles per replicate. Treatments were randomly assigned to cups, and cups were rinsed thoroughly using acetone between experiments. Adult beetles were used only once. The choice of beetles was determined after 6 h by counting the number of beetles inside the cups and represented as percentage choice (percentage of insects responding to either control traps or treatment traps or no response). When two SBA control plugs were tested simultaneously, adult beetles showed no preference for traps on one side of the arena versus the other side. Preliminary experiments showed that the sex of the beetle did not influence the olfactory response towards fungus grown either alone or in the diet enriched with monoterpenes. Therefore, two beetles were randomly chosen for trap bioassays.
For bioassays using terpenes, stock solutions were prepared by dissolving the compounds in DMSO, which were then added to 7% SBA to a final concentration of 0.05 to 1 mg g −1 . To determine the response of adult beetles to (−)-β-pinene and (−)-bornyl acetate amended diet containing the fungus, G. penicillata was used as this species emitted higher amounts of biotransformation products compared to other fungi. Controls were treated with DMSO plus monoterpene (no fungus) or DMSO plus G. penicillata (no monoterpene). ApproximatelyAU : PerPLO 7% SBA plugs (10 mm) supplemented with monoterpenes or plugs containing G. penicillata were placed in the control cups, and G. penicillata-colonized plugs from monoterpene-enriched medium were placed in the treatment cups. The volatile emission from each control and treatment plug used in the bioassays was determined using PDMS tubes as adsorbents and analyzed as described previously (section 1.5). For bioassays with synthetic compounds, stock solutions of authentic standards were prepared by dissolving them in mineral oil (w/v) and further diluted in log 10 steps by dissolving in mineral oil. ApproximatelyAU : PerPLOSstyle; numeralsarenotall 10 μL was applied to 10 mm Whatman filter paper laid on the top of SBA plugs placed inside the cups. Control traps were treated with 10 μL paraffin oil. For the experiment with pheromone blend in the presence of G. penicillata volatiles, G. penicillata-colonized spruce bark plugs were placed in treatment cups, and 10 μL of a pheromone mixture (cis-verbenol:2-methyl-3-buten-2-ol in the ratio of 1:50 diluted 1:100 in paraffin oil) was applied to filter paper as described above. Control cups were treated with 10 μL of the pheromone mixture. Males and females were tested separately in this experiment as I. typographus bark beetles show sex-specific responses to their pheromone [68,114].

Tunneling behavior bioassay
I. typographus tunneling behavior was studied using a protocol modified from [92]. We tested beetles in 35 × 10 mm Petri dishes (Greiner Bio-one, Frickenhausen, Germany) filled with ca. 3 ml of spruce bark diet. The spruce bark diet was prepared as before with some modifications: 7% (w/v) spruce inner bark powder was mixed with 1% fibrous cellulose (Sigma), 2% glucose (Roth), and 4% Bactoagar (Roth) in water and autoclaved for 20 min at 121˚C. Before pouring the medium into the Petri dishes, the medium was mixed with 2% solvent (DMSO: ethanol, 1:1) with 1 mg g −1 of various monoterpenes ((−)-α-pinene, (−)-β-pinene, and (−)-bornyl acetate) and solvent only as a control. For treatment with fungus, 5 μl spore suspension of G. penicillata (1 × 10 6 cells mL −1 ) was added to the center of Petri dishes containing monoterpeneenriched media or solvent controls and incubated at 25˚C for 4 d. A single beetle was introduced per plate, and the plates were sealed with Parafilm and kept in the environmental chamber for 48 h under conditions described above (section 1.2). The beetles were monitored for their tunneling activity after 2, 4, 6, 24, and 48 h with tunneling recorded as a binary event. If beetles were inside the media, it was noted as 1 and, if outside, noted as 0. After 48 h, tunnel lengths made by beetles in each plate were measured using Image J software. Each treatment was replicated with 15 male and female beetles.

Data analysis
IBM SPSS Statistics V25.0 was used to analyze the volatile differences between treatments (E. polonica-, G. penicillata-, L. europhioides-, O. bicolor-, and O. piceae-treated bark samples and untreated control). Data were log-transformed to meet the assumptions of normal distribution, as needed. Concentrations (in dry weight (ng h −1 mg −1 )) of all individual compounds assigned to monoterpenes (MTs) or oxygenated MTs were combined and subjected to a t test or Welch's for estimating differences between control and G. penicillata (Fig 2). Additionally, separate ANOVAs for all individual compounds in each group were also performed (S2 Table). For volatile time course samples, a separate ANOVA test was performed for all individual compounds and compound groups from each fungus with time intervals as an independent factor (S3-S7 Tables). All ANOVA tests were followed by Tukey's post hoc tests to test for differences among treatment combinations. For behavioral bioassays, the CI values from each experimental group were analyzed by Wilcoxon's signed ranked test to compare the differences between control and treatment samples. Binary data from bark beetle tunneling assays were subjected to multiple logistic regression to analyze independent variables such as MT, sex, and fungus that influence the tunneling activity of beetles (dependent variable) in the medium. During data analysis, the male was coded as 1 and female as 0, the presence of fungus coded as 1 and absence of fungus as 0, tunneling inside the medium coded as 1 and not tunneling or staying outside the medium as 0. After testing all possible independent variables and their interactions among them, the following best-fitted logistic regression model was created to predict the odds of beetles tunneling in the different media.
Ln [odds] (tunneling odds) = β0 + β1 � compound + β2 � sex + β3 � fungus Here, β0 is constant, whereas β1, β2, and β3 are logistic coefficients or estimates for the parameters for compound, sex, and fungus, respectively. The strength of association between beetle tunneling odds and effect of MTs or sex or fungus is expressed as odds ratios (OR = exp β ) where OR < 1 indicates a negative relationship between the two events, i.e., the tunneling event is less likely to happen in response to a selected independent variable (coded as 1) in comparison with its base group (coded as 0), OR = 1 indicates no relationship between two events, OR > 1 shows positive relationship between two events.  Table. Principal components (PC1 and PC2) explain 41.4% and 11.2% of the total variation, respectively, and ellipses denote 95% confident intervals around each species. The sPLS-DA plot was generated by using MetaboAnalyst 3.0 software with normalized data (both log transformed and range scaled). The data underlying this Figure Table. Relative amounts (mean ± SE, N = 4-5) of volatiles detected at various time periods after inoculation of fresh spruce bark with E. polonica (4, 8, and 12 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC-MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this  Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with G. penicillata (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC-MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this  Table. Relative amounts (mean ± SE, N = 5) of volatiles detected at various time periods after inoculation of fresh spruce bark with L. europhioides (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC-MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this  O. bicolor (4, 8, 12, and 18 d). Volatiles were collected on polydimethylsiloxane tubes for 2 h and were subjected to GC-MS analysis (see Materials and methods section for details). ND, not detected, NA, not analyzed, TR, trace amounts (<500 TIC counts). The data underlying this  Table. Average colony forming units (CFUs/mL) from untreated, fungus-free, and fungus-free G. penicillata-reinoculated I. typographus bark beetles (n = 5 or 6 beetles). Wash, supernatant from beetles immersed in 0.05% Triton X in 500 μL PBS buffer (pH 7.4); lysate, crushed beetles in 500 μL PBS buffer (pH 7.4); NP, not present. The data underlying this  Table. Colony forming units (CFUs/mL) obtained from bark beetle gallery samples infested by fungus-free beetles, and fungus-free beetles reinoculated with G. penicillata, and untreated control beetles. Approximately 300 mg of bark samples were dissolved in 1 mL PBS buffer solution, and dilutions were plated on PDA. NP, not present. $ Only one gallery sample was tested due to low sample availability.